Any reference to background art herein is not to be construed as an admission that such art constitutes common general knowledge in Australia or elsewhere.
The diaminophenothiazinium dyes are well known. In particular, the medical dye or antidote, methylthioninium chloride (3,7-bis(dimethylamino)phenothiazin-5-ylium chloride), known commonly as “methylene blue”, has in relatively recent times extended its use to a number of medical applications beyond its historic uses. Other common names for this dye include tetramethylthionine chloride, C.I.Solvent Blue, Swiss Blue, C.I.Basic Blue 8, Aniline Violet and Urolene Blue. It has the following structure (Formula I), although a person of skill in the art will appreciate the existence of certain resonance structures and tautomeric forms:

Methylene blue has a long history of varied uses. Industrial applications have included its use as a colouring agent, redox indicator and dye, an immunological or microbiological stain, in photoelectronic imaging, as an environmental metal sequestrant, a leather dye and an antiseptic.
In the clinical field it has two primary established uses: firstly, as an antidote for methaemoglobinaemia, and occasionally for cyanide and carbon monoxide poisoning, and, secondly, as a chromodiagnostic or chromoendoscopic agent for a wide variety of clinical conditions, such as examining cellular dysplasia in for example Barret's Oesphagous and endoscopic polypectomy, and Fallopian tube patency and Fistula detection.
In 1891 Paul Ehrlich identified methylene blue as a treatment for Malaria. More recently it has been suggested that its use may be broadened to include treatment of tauropathies (or neurodegenerative diseases), viral infections, bipolar disorder and tracing of lymph nodes and lymphatic drainage. In dentistry, uses include finding small cracks in teeth and as a photodynamic dye for treating chronic peridontitis. Methylene blue is added to bone cements to provide discrimination between native and synthetic bone. It has also been used as an accelerant to harden bone cement, effectively increasing the speed at which the bone cement can be effectively applied. In species other than human, it has a wide range of uses, from treating “fin rot” in aquarium fish to methaemoglobinaemia in farm dogs caused by their inadvertent ingestion of toxic fox baits.
The original synthesis of methylene blue was developed in Germany in 1877 (German patent No. 1886 to Badische Anilin-und Soda Fabrik) and since that time a number of other methodologies have been described or patented. A common thread to all the methodologies is the use of a range of metal catalysts including salts (or the metals themselves) of iron, manganese, copper, chromium (as chromate), aluminium and zinc, leaving a potential for metal ion contamination from the catalyst. Further metal contamination may come from the types of metal equipment used for the synthetic processes.
In addition to metal residues, the chemistry of methylene blue lends itself, during production, to the synthesis or inter-conversion of three other structurally and chemically similar organic entities. While these may have little effect on the industrial uses of methylene blue, and may also be used for similar cytological staining purposes, in clinical use these may be considered as undesired inclusions and are specifically cited in the United States Pharmacopoeia (USP) and British/European Pharmacopoeia (BP/EP) as contaminants.
These organic contaminants are collectively called “Azures” and come from some level of demethylation of the two dimethylamino groups at the 3 and 5 positions on the ring structure of methylene blue. Specifically they are: the trimethyl derivative known as Azure B (FORMULA II); the dimethyl derivative known as Azure A (FORMULA III) and monomethyl derivative known as Azure C (FORMULA IV).

Due to the structural and chemical similarity of the Azures to methylene blue, these ‘contaminants’ are difficult to separate or remove from a mixture by standard means. It has also proven to be difficult to reduce their occurrence during synthesis of methylene blue. Nevertheless, numerous attempts have been made to remove or reduce the levels of Azures during the synthetic process, including re-purification by many means including recrystallisation of the final methylene blue product itself. Marshall and Lewis (1975) describe the purification of commercial methylene blue and Azure B by solvent extraction at a high pH of 9.5 with carbon tetrachloride and subsequent recrystallisation. They also describe metal ion removal by low temperature, low pH crystallisation. Lohr et al (1975) describe a purification process utilising column ion chromatography which is not practicable on a commercial scale.
More recently, in 2005 Storey et al (WO 2006/032879) described processes for the de novo manufacture and purification of methylene blue and derivatives thereof using a number of metal ions as catalysts within stepped processes (including chromate (IV) and copper (II) sulphate and iron (II) oxide) under controlled pH and temperature conditions. This is followed by washing/solvent extraction with organic solvents such as dichloromethane, 1,2-dichloroethane, chloroform, ethyl acetate, diethyl ether, chlorobenzene, petroleum ether, benzene, toluene and methyl acetate. Key steps in the final stage of the process include the addition of dimethyldithiocarbamate (DT), a sulphide, and a chloride salt such as sodium chloride, and carbonate such as sodium carbonate followed again by organic solvent washing and the addition of EDTA (ethylenediaminetetraacetic acid), followed again by organic solvent washing and recrystallisation and washing at a low pH in the presence of an organic solvent such as dichloromethane or tetrahydrofuran.
Further work by the same group (WO 2008/007074) describes purification of methylene blue by acyl derivatisation, at the N10 position (or a number of other organic derivatives including saturated aliphatic derivatives) and purification (with agents such as activated charcoal) and conversion by oxidation to the original methylene blue. The document also includes the concept of acylating a methylene blue precursor and purifying via the same processes.
A number of authors, in a similar fashion, have postulated that metal and organic residue removal may be achieved by further derivatisation of the methylene blue, post manufacture, so as to increase the chemical differences between the Azures and methylene blue and also to allow ease of metal residue removal. Buc et al 1959 (U.S. Pat. No. 2,909,520) described processes for the manufacture of acylated leuco methylene blue, in particular benzoyl leuco methylene blue.
Gensler et al, 1966, describes the simple oxidative re-conversion of N-benzoyl leuco methylene blue to methylene blue. In fact all that was required was the presence of oxygen for an autoconversion.
Feraud et al (WO2008/006979) describe a method for industrial purification of methylene blue and other like compounds where both the Azures and metal levels are alleged to be reduced based on the formation of a large organic derivative of methylene blue by reaction to form a N—C bond at the N10 position of a diaminophenothiazinium compound.
This complex multi-step derivatisation of methylene blue (or a related derivative thereof) begins with reduction of the N followed by reaction of the resulting amine with certain derivative options. They then proceed to describe standard methods and/or concepts for purification of the complex organic derivative, including reduction of metal ion levels via filtration of the derivatised organic material through a support that retains metals, crystallisation from an appropriate solvent and other known methods. They use solvent washing or recrystallisation to reduce the Azure levels in this derivative. Again, the final process is reconversion or oxidation to methylene blue using quinones, nitric acid, perchloric acid iodine, hydrochloric acid, sulphuric acid, hydrogen peroxide or UV light, with a preference for 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).
In summary, the levels of metals and/or organic impurities found within methylene blue samples is not unexpected when such historic synthetic approaches are considered, and may even be acceptable for the commercial product in use for many industrial and cytological purposes where these potential residues do not affect the use. However, with recent requirements for lower levels of these impurities in methylene blue used for pharmaceutical purposes, it has become necessary to address the removal of impurities in an efficient, simple and cost effective purification process.